Advances in identifying the mechanisms that control lung cell differentiation in development have also enhanced the field of lung regeneration and repair since many of the developmental pathways get reactivated or silenced during lung injury or in disease. The discovery that more than 90% of the genome is transcribed into RNAs with no protein coding ability (ncRNAs) has uncovered thousands of RNAs with potential regulatory functions. Among them, long ncRNAs (>200 nt, lcnRNAs) are expressed in highly specific patterns, are regulated in development, and altered in disease. To date, the functions of most of the lncRNAs identified in the lung are uncharacterized. A large number of lncRNAs originate by divergent transcription at promoters of genes encoding transcription factors and developmental genes. The transcription factors GATA6 and NKX2-1 are critical for endoderm and lung epithelial cell differentiation in humans and mice. The lncRNA transcripts GATA6-AS1 and NKX2-1-AS1, divergent to these transcription factors, follow similar patterns of expression to GATA6 and NKX2-1 respectively. Our initial studies indicate that Gata6-AS1 controls Gata6 expression in cis likely by its interaction with the histone3-lysine-27 demethylase KDM6a, suggesting a role in epigenetic regulation. NKX2-1-AS1 on the contrary, does not control NKX2-1 expression; rather it regulates genes in trans and localizes to the nucleolus or the cytoplasm (away from active transcription), suggesting a role in post-transcriptional regulation. We hypothesize that the human GATA6-AS1 and NKX2-1-AS1 have distinct and essential roles in lung development by regulating epithelial cell differentiation. To study their functions we will us a unique in vitro system of human induced pluripotent stem cells (iPSCs) recently developed in our labs that recapitulates the early stages of human endoderm and lung development. This system allows producing a high number of human cells representing definitive endoderm, lung epithelial progenitors and more mature lung epithelial cells for functional and molecular studies that usually require a high number of cells. In Aim 1 we will test whether GATA6-AS1 enhances endoderm commitment in human pluripotent stem cells by recruiting chromatin activating proteins in cis, and in Aim2, whether NKX2-1AS promotes endoderm-to-lung epithelial cell differentiation by post-transcriptional regulation of gene expression in trans. In Aim3 we will compare functional conservation of the lncRNAs in mice and humans and whether they are necessary for in vivo mouse endoderm and lung development. lncRNA loss- or gain-of-function experiments in iPSCs will inform us about the role of these lncRNAs in endoderm specification and lung cell differentiation, limiting alternative endoderm-derived cell lineages. Optimal development of the respiratory system is essential to health at birth and beyond. Our findings will guide future studies about lncRNA mediated pathways that may become new targets for disease treatment, or new markers to follow in the diagnosis of developmental or adult pulmonary diseases.